Inhibition of Antigen Presentation with Poorly Catabolized Polymers

ABSTRACT

Methods to prevent the rejection of immunogenic tissues in an animal by administering a non-immunogenic, poorly catabolized molecule in an amount sufficient to inhibit an immune response are described herein. Also described are compositions that are useful for inhibiting immune responses in animals that are recipients of cellular transplants. For example, these methods and compositions can be used to prevent the rejection of xenografted and allografted tissues in an animal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Application Ser.No. 12/463,315 filed May 8, 2009, now issued as U.S. Pat. No. 7,786,096;which is a divisional application of U.S. application Ser. No.10/381,855 filed Oct. 8, 2003, now issued as U.S. Pat. No. 7,538,097;which is a 35 USC §371 National Stage application of InternationalApplication No. PCT/US01/42329 filed Sep. 25, 2001; which claims thebenefit under 35 USC §119(e) to U.S. Application Ser. No. 60/235,321filed Sep. 26, 2000, now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF INVENTION

1. Field of the Invention

The present invention relates to the field of immunosuppression, morespecifically to methods of inhibiting antigen presentation andtransplant rejection.

2. Background Information

A number of diseases are treated by the transplantation of tissuedonated by other humans (allografts) or obtained from animals(xenografts). For example, insulin-dependent diabetes is often treatedby transplantation of insulin-secreting pancreatic islet cells. Whilethe transplanted cells may have the capacity to perform the desiredfunction (e.g., secretion of insulin in response to rising levels ofglucose), such grafts typically fail as a result of immunologicalrejection. Shortly after transplantation, cells of the immune system ofthe recipient recognize the allogeneic or xenogeneic cells as foreignand proceed to attack the graft through both humoral and cellularroutes. Allogeneic or xenogeneic cells are initially recognized by therecipient's immune system through antigenic determinants expressed onthe surface of the cells. The predominant antigens recognized as“non-self” are major histocompatibility complex class I and class IIantigens (MHC class I and class II). MHC class I antigens are expressedon virtually all parenchymal cells (e.g., pancreatic islet cells), incontrast, MHC class II antigens are expressed on a limited number ofcell types, primarily B cells, macrophages, dendritic cells, Langerhanscells and thymic epithelium. The interaction of foreign MHC antigenswith the T cell receptor on host T cells causes these cells to becomeactivated. Following activation, the T cells proliferate and induceeffector functions which result in cell lysis and destruction of thetransplanted cells.

For transplantation to be a viable therapeutic option, approaches areneeded to inhibit rejection of transplanted cells by the immune systemof the recipient. One method for inhibiting this rejection process is byadministration of drugs that suppress the function of the immune system.While drugs such as cyclophosphamide and cyclosporin effectively inhibitthe actions of the immune system and thus allow graft acceptance, theiruse can cause generalized, non-specific immunosuppression in the graftrecipient which leaves the recipient susceptible to other disorders suchas infection and tumor growth. Additionally, administration ofimmunosuppressive drugs is often accompanied by other serious sideeffects such as renal failure and hypertension.

It has been shown that it is possible to alter an antigen on the surfaceof a cell prior to transplantation to “mask” the antigen from normalrecognition by cells of the recipient's immune system (see, Faustman andCoe, Science 252:1700-1702 (1991) and WO 92/04033). For example, MHCclass I antigens on transplanted cells can be altered by contacting thecells with a molecule which binds to the antigen, such as an antibody orfragment thereof (e.g., a F(ab′)₂ fragment) prior to transplantation.This alteration of MHC class I antigens modifies the interaction betweenthe antigens on the cells and T lymphocytes in the recipient followingtransplantation to thereby inhibit rejection of the transplanted cells.Additional methods for reducing the immunogenicity of an allograft orxenograft to inhibit rejection of the graft following transplantation ina host are needed.

T-cell mediated immune responses are thought to be the primary mechanismof organ transplant rejection and a driving component of variousauto-immune diseases. This T-cell mediated immune response is initiallytriggered by helper T-cells which are capable of recognizing specificantigens. These helper T-cells may be memory cells left over from aprevious immune response or naive cells which are released by the thymusand may have any of an extremely wide variety of antigen receptors. Whenone of these helper T-cells recognizes an antigen present on the surfaceof an antigen presenting cell (APC) or a macrophage in the form of anantigen-MHC complex, the helper T-cell is stimulated by signalsemanating from the antigen-specific T-cell receptor, co-receptors, andIL-1 secreted by the APC or macrophage, to produce IL-2. The helperT-cells then proliferate. Proliferation results in a large population ofT-cells which are clonally selected to recognize a particular antigen.T-cell activation may also stimulate B-cell activation and nonspecificmacrophage responses. Some of these proliferating cells differentiateinto cytotoxic T-cells which destroy cells having the selected antigen.After the antigen is no longer present, the mature clonally selectedcells will remain as memory helper and memory cytotoxic T-cells, whichwill circulate in the body and recognize the antigen should it show upagain. If the antigen triggering this response is not a foreign antigen,but a self antigen, the result is auto immune disease; if the antigen isan antigen from a transplanted organ, the result is graft rejection.Consequently, it is desirable to be able to regulate this T cellmediated immune response.

The current paradigms of immunosuppressive agents reflects the progressin understanding the cellular and molecular mechanisms which mediategraft rejection. Six paradigms represent the evolution ofimmunosuppressive strategies for organ transplantation to date. Theproliferation paradigm advances agents which interrupt lymphocyte celldivision (azathioprine, cyclophosphamide, mycophenolic acid). Thedepletion paradigm conscripts drugs that bind to lymphocyte cell surfacemarkers, thereby producing cell lysis and/or inactivation (polyclonalATGAM and thymoglobulin, and monoclonal OKT3 antilymphocyte antibodies).The cytokine paradigm uses agents that interrupt lymphocyte maturationalevents; eg, synthesis (calcineurin inhibitors; cyclosporine/tacrolimus),binding to surface receptors (anti-CD25 mAbs), or signal transductionphases of cytokine stimulation (sirolimus). The introduction ofcalcineurin inhibitors markedly reduces the rate of acute rejectionepisodes and increases short-term graft survival rates; nephrotoxicityand chronic allograft attrition remain as unanswered challenges. Thecyclosporine A (CsA) sparing property of sirolimus permits the use oflower exposure to calcineurin agents, allows for early withdrawal ofsteroid therapy, and may delay allograft senescence. Furthermore, thecombination of SRL with anti-IL-2R mAbs proffers an induction approachwhich allows prolonged periods of holiday from calcineurin inhibitors.To address the tissue nonselectivity of the calcineurin and mTORinhibitors, which presumably causes the drug toxicities, new agents arebeing developed to selectively inhibit the T cell target Janus Kinase 3.In the costimulation paradigm, the accessory signals generated byantigen-presenting cells are interrupted by distinct agents: thereceptor conjugate CTLA4-immunoglobulin and anti-B7 or anti-CD40 ligandmAbs. Another set of drugs (selectin blocking agents, anti-ICAM-1antisense deoxy oligonucleotides, and the lymphocyte homing inhibitorFTY720) seeks to modulate the ischemia-reperfusion injury, whichexacerbates cytokine-mediated events in the donor and the subsequentprocurement injury and may also accelerate the progression of transplantsenescence. Finally, the transplantation tolerance paradigm is based onthe development of strategies which distort alloimmune recognition byantigen reactive cells (MHC peptides or proteins), produce anergy(costimulation blockers), functional inactivation, or deletion ofantigen-reactive cells (donor bone marrow infusions and gene therapy).

Thus, the common paradigms today focus upon either T-cell expansion orextravasation into the rejected tissue site. However, a relativelyignored component of immune rejection is antigen presentation, which wenow document herein as an excellent target for intervention through theuse of poorly catabolized polymers.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that foreign antigenpresentation can be inhibited in an animal by saturating the antigenpresenting cells (APC's) with non immunogenic agents. In general,methods are provided to saturate antigen-presenting cells with poorlycatabolized non immunogenic-polymers that are readily phagocytosed byAPC's such that the presentation of immunogenic foreign antigens areeffectively inhibited.

One embodiment of the present invention provides a method of inhibitingantigen presentation in an animal by administering to an animal a poorlycatabolized polymer in an amount sufficient to inhibit presentation ofat least one antigen to the immune system of the animal. The at leastone antigen can be derived from a variety of sources such as, but notlimited to, allografted cells, xenografted cells, isolated stem cellsand gene therapy formulations. Furthermore, the at least one antigen canbe derived from a source that is substantially free of nucleic acid.

Another embodiment of this invention provides a method of inhibiting therejection of cells transplanted in animals by administering a poorlycatabolized polymer to an animal then introducing, into the animal,cells that are capable of expressing at least one antigen. Cells thatare capable of expressing at least one antigen can be but are notlimited to allografted cell, xenografted cells and isolated stem cells.

Yet another embodiment of the present invention provides a method ofinhibiting an immune response to a gene therapy formulation byadministering a poorly catabolized polymer to an animal thenintroducing, into the animal, a gene therapy formulation that is capableof producing at least one antigen.

In each of the previously described methods, the time of administrationof the poorly catabolized polymer can be altered. Specifically, thepoorly catabolized polymer can be administered to the animal before atleast one antigen is presented the immune system of the animal. Thepoorly catabolized polymer can also be administered to the animal beforeexposing the animal to at least one antigen. Even more specifically, thepoorly catabolized polymer is administered to the animal more than 24hours prior to exposing the animal to at least one antigen.

Other embodiments of this invention describe administration of thepoorly catabolized polymer to an animal in the presence of otherimmunosuppressive agents. In still other embodiments the poorlycatabolized polymer comprises a dextran solution.

The present invention also provides a pharmaceutical compositioncomprising a poorly catabolized polymer wherein the poorly catabolizedpolymer is used to inhibit the presentation of at least one antigen tothe immune system of an animal. In some of these compositions, thepoorly catabolized polymer comprises a dextran solution.

Still another embodiment of the present invention provides a use of apoorly catabolized polymer in the preparation of a medicament fortreating rejection by the immune system of an antigenic materialintroduced into an animal. This antigenic material can comprise a cell.It can also comprise a gene therapy formulation. In some uses, thepoorly catabolized polymer is dextran.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a schematic which illustrates the chemical structure of aportion of a dextran polymer.

FIG. 2 is a graph which shows the effect of pre-administration of adextran solution on the survival of human HT1080 cell xenografts thatare grown inside titanium chambers which have been implanted in C57BI/6mice.

FIG. 3 is a graph which shows the effect of the route of dextranadministration on the survival of human HT1080 cell xenografts that aregrown inside titanium chambers which have been implanted in C57BI/6mice.

FIG. 4 is a graph which illustrates the effect of the time ofadministration of dextran on the survival of human HT1080 cellxenografts that are grown inside titanium chambers which have beenimplanted in C57BI/6 mice.

FIG. 5 is a graph which plots the survival of Murine NOD-derived betastem cell isografts in immunocompatible C57BI/6 mice.

FIG. 6 is a graph which plots the survival of a Murine NOD-derived betastem cell allograft in Balb/c mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Dextran is a polymer of anhydroglucose. FIG. 1 shows the unit structureof a typical a polymer of dextran. Approximately 95% of the dextranpolymer is composed of D-glucose molecules having α(1→6) linkages(Rankinet et al., J. Am. Chem. Soc. 76:4435 (1954)). The remaining 5% iscomposed of glucose molecules linked together by α(1→6) glycosidicbonds. These α(1→6) linkages account for the branching of the dextranpolymer. Conflicting data on the branch lengths implies that the averagebranch length is less than three glucose units. However, other methodsindicate branches of greater than 50 glucose units exist.

The molecular weight of a dextran polymer affects its structure. Nativedextran has been found to have a molecular weight (MW) in the range of 9million to 500 million Daltons (Da). This molecular weight rangecorresponds roughly to dextrans having between 50,000 and 2.8 millionglucose molecules. Many of the more commonly used dextrans are of lowerMW than the native polymers. These lower MW dextrans exhibit slightlyless branching and have a more narrow range of MW distribution than thenative polymers. Dextrans with MW greater than 10,000 glucose moleculesbehave as if they are highly branched. However, as the MW increases,dextran molecules attain greater symmetry. Dextrans with MW of 2,000 to10,000 glucose molecules exhibit the properties of an expandable coil.At MWs below 2,000 glucose molecules, dextran is more rod-like.

There are a variety of techniques that are commonly used to determinethe MW of dextran polymers. For example, the MW of dextran can bemeasured by one or more of the following methods: low angle laser lightscattering, size exclusion chromatography, copper-complexation andanthrone reagent colorometric reducing-end sugar determination andviscosity.

Most dextrans are derived from Leuconostoc mesenteroides, strain B 512.Shorter dextran polymers of various MWs are then produced by limitedhydrolysis and fractionation although exact methods are heldproprietary. In general, however, fractionation of these polymers can beaccomplished by size exclusion chromatography or ethanol fractionationin which the largest MW dextrans precipitate first.

Pharmaceutically acceptable compositions contemplated for use in thepractice of the present invention can be used in the form of a solid, asolution, an emulsion, a dispersion, a micelle, a liposome, and thelike, wherein the resulting composition contains one or more of theactive compounds contemplated for use herein, as active ingredientsthereof, in admixture with an organic or inorganic carrier or excipientsuitable for nasal, enteral or parenteral applications. The activeingredients may be compounded, for example, with the usual non-toxic,pharmaceutically or physiologically acceptable carriers for tablets,pellets, capsules, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, suppositories, solutions, emulsions,suspensions, hard or soft capsules, caplets or syrups or elixirs and anyother form suitable for use. In addition auxiliary, stabilizing,thickening and coloring agents may be used. The active compoundscontemplated for use herein are included in the pharmaceuticalcomposition in an amount sufficient to produce the desired effect uponthe target process, condition or disease.

In addition, such compositions may contain one or more agents selectedfrom flavoring agents (such as peppermint oil of wintergreen or cherry),coloring agents, preserving agents, and the like, in order to providepharmaceutically elegant and palatable preparations. Tablets containingthe active ingredients in admixture with non-toxic pharmaceuticallyacceptable excipients may also be manufactured by known methods. Theexcipients used may be, for example, (1) inert diluents, such as calciumcarbonate, lactose, calcium phosphate, sodium phosphate, and the like;(2) granulating and disintegrating agents, such as com starch, potatostarch, alginic acid, and the like; (3) binding agents, such as gumtragacanth, com starch, gelatin, acacia, and the like; and (4)lubricating agents, such as magnesium stearate, stearic acid, talc, andthe tike. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract, thereby providing sustained action over a longerperiod. For example, a time delay material such as glyceryl monostearateor glyceryl distearate may be employed. The tablets may also be coatedby the techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452;and 4,265,874 to form osmotic therapeutic tablets for controlledrelease.

When formulations for oral use are in the form of hard gelatin capsules,the active ingredients may be mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin, or the like. Theymay also be in the form of soft gelatin capsules wherein the activeingredients are mixed with water or an oil medium, for example, peanutoil, liquid paraffin, olive oil and the like.

Formulations may also be in the form of a sterile injectable suspension.Such a suspension may be formulated according to known methods usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,4-butanediol. Sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides, fatty acids (including oleic acid), naturally occurringvegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil,etc., or synthetic fatty vehicles like ethyl oleate or the like.Buffers, preservatives, antioxidants, and the like can be incorporatedas required.

Formulations contemplated for use in the practice of the presentinvention may also be administered in the form of suppositories forrectal administration of the active ingredients. These compositions maybe prepared by mixing the active ingredients with a suitablenon-irritating excipient, such as cocoa butter, synthetic glycerideesters of polyethylene glycols (which are solid at ordinarytemperatures, but liquify and/or dissolve in the rectal cavity torelease the active ingredients), and the like.

In addition, sustained release systems, including semi-permeable polymermatrices in the form of shaped articles (e.g., films or microcapsules)can also be used for the administration of the active compound employedherein. The poorly catabolized polymer can also be provided as a unitdosage such as a septum-sealed vial, either lyophilized or in aqueoussolution.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the invention. The examples are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to the numbers disclosed herein(e.g. amounts, temperatures, etc.); however, those skilled in the artwill account for some experimental error and deviation. Unless indicatedotherwise, molecular weights are reported as average molecular weight.

Example 1 Inhibition of Xenograft Tissue Rejection Through Pre-Treatmentwith a Dextran Polymer

Dextran, derived from Leuconostoc mesenteroides, strain B512 (averageMolecular Weight 500,000 Da) was used in the following studies. Adextran solution, suitable for administration to animals, was made bydissolving solid dextran in sterile deionized water (dH₂O) to a finalconcentration of 10%.

For cellular implants into mice, human HT1080 spheroids expressing greenfluorescent protein were used. These spheroids were prepared using thefollowing method. Histone H2B-GFP, prepared as previously described(Kanda, et al., Curr. Biol. 26:377-85 (1998)), was subcloned into theLXRN retroviral vector (Clontech, Palo Alto Calif.). The resultantH2B-GFP LXRN vector was cotransfected with VSVG into GP-293 cells(Clontech) and viral supernatants harvested 48 hours post transaction.Retroviral supernatants were concentrated by centrifugation at 50,000 gand stored at −80° C. until use. HT1080 cells (obtained from ATCC) orIPSC (pancreatic beta stem cells (provided by Ixlon Biotechnology,Alachua, Fla.)) were transduced with VSVG pseudotyped H2BGFP LXRN virusstocks for 48 hours with 5 μg/ml polybrene and were selected in 300μg/ml G418 for 2 weeks. Pooled cells that expressed H2BGFP, asdetermined by fluorescent microscopy and FACs analysis, were expandedand used for in vivo experiments. HT1080 cells were passaged in DMEM 4.5g/L glucose supplemented with pyruvate, glutamine, non-essential aminoacids, and gentamicin (50 μg/ml) and maintained in a humidified 5% CO₂atmosphere at 37° C. Cells were routinely tested for mycoplasmacontamination with the Genprobe mycoplasma detection kit. Suspensions oftrypsinized monolayers were washed with fresh complete medium viabilitytested with trypan blue exclusion, and diluted to a final volume of250,000 cells/ml. The cell suspensions were dispersed 100 ul/well into96 well round bottom plates coated with 1.0% agarose for a liquidoverlay. The spheroids were allowed to compact for 48 hours followed bywashing in serum free media for implantation into mice bearing titaniumchambers.

C57B1/6 mice were prepared by surgically implanting titanium chambersinto a dorsal skinfold as described previously, (see, Lehr, et al., Am.J. Pathol. 143:1055-1062 (1993); Torres et. al., Microvascular Research49:212-226 (1995)). In brief, male mice (25-35 g body weight) wereanesthetized (7.3 mg ketamine hydrochloride and 2.3 mg xylazine/100 gbody weight, i.p.) and placed on a heating pad. Two symmetrical titaniumframes were implanted into a dorsal skinfold, so as to sandwich theextended double layer of skin. A 15 mm full thickness skin layer wasexcised. The underlying muscle (M. cutaneous max.) and subcutaneoustissues were covered with a glass cover slip incorporated in one of theframes.

After a recovery period of 2-5 days, the mice were divided into bothtreatment and control groups. A 200 μl injection of the sterile 10%dextran solution was administered to the treatment group intravenouslythrough the tail vein 48 hours prior to spheroid implantation.Equivalent injections of dH₂O were administered to control group mice. Asecond 200 μl injection of the sterile 10% dextran solution wasadministered to the treatment group 24 hours after the first injection,whereas the control group received dH₂O. On the day of implantation, anequivalent number of HT1080 spheroid cells expressing green fluorescentprotein were implanted into the titanium chambers of both the controland treatment group mice. Subsequent to spheroid implantation, and forthe duration of the experiment, 100 μl of the 10% sterile dextransolution was administered to each mouse in the treatment groupintravenously through the tail vein at 24 hour intervals. Equivalentinjections of dH₂O were administered to the control mice. Throughout thecourse of the experiment, the size of HT1080 cell xenografts weremeasured by fluorescent intravital microscopy. This microscopy wasperformed using a Mikron Instrument Microscope (Mikron Instrument, SanDiego, Calif.) equipped with epi-illuminator and video-triggeredstroboscope illumination from a xenon arc (MV-7600, EG&G, Salem, Mass.).A silicon intensified target camera (SIT68, Dage-MTI, Michigan City,Ind.) was attached to the microscope. A Hamamatsu image processor (Argus20) with firmware version 2.50 (Hamamatsu Photonic System, USA) was usedfor image enhancement and to capture images to a computer. A LeitzPL1/0.04 objective was used to obtain an over-view of the chamber andfor determination of graft size.

Statistical analysis was made using a statistical software package(SigmaStat, Jandel Scientific). Statistical analysis was made using bothanalysis of variance and multiple comparison tests. For all tests, pvalues smaller than 5% were considered significant. Data was presentedas MEAN±STD.

FIG. 2 plots the survival of the HT1080 spheroid xenografts for bothdextran-treated mice and the control group. For the mice treated withdextran, the size of the HT 1080 xenograft increases throughout thecourse of the experiment. By the end of the experiment, on day 14, thesize of the xenograft has more than doubled. By contrast, the xenograftsin the control mice decrease throughout the course of the experiment andhave been eliminated by day 14. These results indicate that pretreatmentwith 10% dextran solution for 48 hours prior to transplantation resultsin effective inhibition of xenograft rejection.

Example 2 Effects of the Route of Administration of the Dextran Polymeron Xenograft Survival

To examine the temporal and spatial dependence of the dextran polymer ongraft survival, dextran was administered intraperitoneally (i.p.) versesintraveneously (i.v.). The methodology of Example 1 was used with thefollowing modifications. C57B1/6 mice having implanted titanium chamberswere divided into four groups. The first group was designated as thecontrol group and received no treatment. The second group received 200μl i.p. injections of the sterile 10% dextran solution every 24 hoursbeginning two days prior to spheroid implantation. On the day ofspheroid implantation and thereafter, the injection volume was reducedto 100 μl. The third group of mice received 100 μl i.v. injections ofthe sterile 10% dextran solution every 24 hours beginning four daysafter spheroid implantation. The fourth group (designated the Re-Implantgroup) was comprised of mice that had rejected a spheroid xenograft thathad been implanted 10 previously. This group received i.v. dextrantreatments beginning six day prior to re-implantation of spheroids. Onthe first and second day of treatment, a 200 μl volume of the steriledextran solution was administered. On each day thereafter, the volumewas decreased to 100 μl. These 100 μl injections were continuedthroughout the course of the experiment.

FIG. 3 shows the xenograft survival over the 14 day course of theexperiment for each group of mice. By day 14, every group hasexperienced a significant reduction in xenograft size. In contrast withthe i.v. dextran pretreatments described in Example 1, i.p.administration of dextran beginning 48 hours prior to implantation(group 2) did not enhance the survival of xenografts. Similarly,xenograft survival was not enhanced by i.v. treatments commencing fourdays after spheroid implantation (group 3). This result suggests thatthe mechanism by which dextran acts is not through inhibition of theability of T-cells to extravasate into the chamber.

FIG. 3 also shows that dextran pretreatment could not protect spheroidswhich had been implanted into mice that had previously rejected aspheroid graft (group 4). By day 10 of the experiment, the spheroidgraft was eliminated. Accordingly, this result eliminates thepossibility of direct T-cell inhibition as a mechanism of suppression.

Example 3 The Effect of Dextran Uptake on Antigen Presenting Cells

The uptake of dextran by APCs was shown by injecting mice withfluorescein isothiocyanate (FITC) labeled dextran then visualizingtissue sections by fluorescent microscopy. C57/B16 mice were dividedinto two groups. Mice in the first group received a 200 μl i.v.injection of a 2% FITG labeled dextran solution (average MW of dextran500,000 Da). Twenty-four hours later, the animals were sacrificed andorgan sections were whole mounted and imaged using fluorescentmicroscopy. Mice in the second group received a 200 μl i.v. injection ofan unlabeled 10% dextran solution one daily for 48 hours. At the end ofthe 48 hour period, the mice were given a 200 μl i.v. injection of the2% FITC labeled dextran solution. Twenty-four hours later, these animalswere subjected to the same treatment as mice in the first group.

Analysis of the tissues of mice in the first group revealedmacrophage-like cells taking up the labeled dextran polymer in alltissues examined including brain, lung, spleen, kidney, peritoneum,lymph-nodes, skin, and liver. Analysis of the tissues of mice in thesecond group revealed no labeling which indicated that saturation ofthese cells with the unlabeled polymer had occurred. The conclusion fromthese studies was that perturbation of antigen presenting cell functionwas the principal mechanism by which this poorly catabolized polymerblocked transplant rejection.

Example 4 Temporal Optimization of Dextran Administration for theSurvival of Xenografts in Mice

To demonstrate the temporal effect of dextran administration onxenografts, the methodology of Example 1 was used with the followingmodifications. C57B1/6 mice having implanted titanium chambers weredivided into three groups. The first group was designated as the controlgroup and received no treatment. The second group received 200 μl i.v.injections of the sterile 10% dextran solution every 24 hours beginningtwo days prior to spheroid implantation. On the day of spheroidimplantation and thereafter, the injection volume was reduced to 100 μl.The third group received a 200 μl i.v. injection of the sterile 10%dextran solution 24 hours prior to spheroid implantation. On the day ofspheroid implantation and every 24 hours thereafter, 100 μl injectionswere given.

FIG. 4 shows the effect of the length of dextran pretreatment on thesurvival of xenografts. Twenty four hour pretreatment with dextran onlyslightly increases the survival of the xenograft relative to the controlBy contrast, when treatment is started 48 hours prior to spheroidimplantation, the xenograft survival is greatly enhanced. The resultspresented in FIG. 4 together with those in Example 3 show that completesaturation of the APCs is required for effective inhibition of graftrejection.

Example 5 Inhibition of Allograft Stem Cell Rejection by DextranPretreatment

Allograft stem cells transplants were tested to examine if such cellswould benefit from systemically administered poorly catabolizedpolymers. The methodology of Example 1 was used with the followingmodification. Beta stem cells from the pancreas derived from NOD micestably transfected with green fluorescent protein, prepared aspreviously described (Ramiya et al, Nature Medicine 6:278-282 (2000)),were implanted into the chambers of C57BL/6 mice or Balb/c mice.

FIG. 5 plots the survival of beta stem cell grafts in C57BL/6 mice forboth the control and treatment groups. A similar increase in graft sizefor both the control and dextran-treated mice is shown throughout thecourse of the experiment. These results indicate that systemicpretreatment with dextran had no significant effect on the growth of thebeta stem cells spheroids that were grown as isografts in the chamber ofC57BL/6 mice.

By contrast, a significant difference in graft survival between thecontrol and treatment groups for Balb/c mice can be observed. FIG. 6shows that beta stem cell allografts fail to survive in untreatedcontrol mice by the end of the 35 day experiment. The beta stem cellallografts of the dextran-treated mice, however, show significantincrease in size over the course of the experiment with a greater than40-fold increase on day 35. These results and those from the previousexamples demonstrate that both allografts and xenografts are protectedby pre-administration with poorly catabolized polymers.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit andscope of that which is described and claimed.

1. A pharmaceutical composition comprising a poorly catabolized polymerwherein said poorly catabolized polymer is used to inhibit thepresentation of at least one antigen to the immune system of an animal.2. The pharmaceutical composition of claim 1, wherein said poorlycatabolized polymer comprises a dextran solution.
 3. The pharmaceuticalcomposition of claim 1, wherein the antigen comprises a cell.
 4. Thepharmaceutical composition of claim 1, wherein said poorly catabolizedpolymer is dextran.